PROJECT SUMMARY Epigenetic changes are a result of the activity of DNA methyltransferases and histone readerss, writers and erasers. One such histone writer is EZH2, a key player in regulating genes involved in differentiation, development, and stem cell pluripotency. EZH2 is a histone methyltransferase comprised of four domains: Domain I, Domain II, CXC domain, and the catalytic SET domain. EZH2 is a subunit of PRC2 that, together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12), catalyzes the sequential mono-, di- and trimethylation of H3K27, an epigenetic mark associated with gene silencing. It is now widely recognized that histone modifications participate in a complex network of epigenetic crosstalk. As histone PTMs are reversible and dynamic in nature, the proteins that modify these crosstalk networks are sought after as therapeutic targets for epigenetic reprogramming. We propose that EZH2 misregulation imparts changes in the chromatin modification landscape brought about by its interactions with other chromatin readers, writers and/or erasers and downstream expression of other Chromatin Regulatory Factors (CRF). To test this hypothesis, we will use isogenic cell lines created by stable transfection to explore the resultant changes in the chromatin landscape brought about by EZH2 mutations. Proteomics will be used to determine changes in histone modifications and EZH2 interactors. Next RNA-Seq we be used to determine changes in CRFs tied to observed histone modification changes. And finally, we will perform CHIP-Seq to determine occupancy of EZH2 and aberrant CRFs at cancer genes and other loci. These data will fill a gap in knowledge as to how EZH2 alters the chromatin landscape and leads to cellular transformation.